The present invention relates to novel microcapsules having an anisotropic salt membrane encapsulating an aqueous or substantially aqueous core which may comprise various active agents. The microcapsules are prepared by the interfacial reaction, in aqueous medium, of Lewis acid and base wall-forming reactants.
Microencapsulation is a process by which a relatively thin coating can be applied to dispersions of small particles of solids or droplets of liquids, thus providing a means for converting liquids to solids, altering colloidal and surface properties, providing environmental protection, and controlling the release characteristics or availability of coated materials. Several of these properties can be attained by macropackaging techniques; however, the uniqueness of microencapsulation is the smallness of the coated particles and their subsequent use and adaptation to a wide variety of dosage forms and product applications. Heretofore, known feasible methods for producing microcapsules on an industrial scale have often involved the use of organic solvents. However, the use of organic solvents may present environmental and safety problems. In addition, it is often difficult to remove all the organic solvent from the microcapsules, thus leaving organic contaminants.
It has been proposed to use microcapsules as a means of delivering vaccine. Two broad types of antigen delivery systems have been studied for their capacity to enhance immunity: solid (or porous) microcapsules and microcapsules with a core region surrounded by a physically distinct wall. Solid is microcapsules may be prepared by a variety of processes including coacervation of colloids (Kwok, K. K., et al., 1991, Pharm. Res., 8: 341-344), precipitation of proteins by physical means (e.g., phase separation) or chemical agents (e.g., acid chlorides) (Levy, M. C., et al., 1991, J. Pharm. Sci., 80: 578-585), or solvent evaporation techniques that surround aqueous dispersions with polyester films (Singh, M. et al., 1991, Pharm. Res., 8: 958-961). Wall/core systems shown useful for antigen delivery include liposomes (Gerlier, D. et al., 1983, J. Immunol., 131: 490), ISCOMS (Claasen, I., and Osterhaus, A., 1992, Res. Immunol., 143: 531-541) and proteosomes (Gould-Fogerite, S. and Mannino, R., 1992, Liposome Technology, Volume III, Gregoriadis, G., (ed.), CRC Press, Boca Ration, Fla.; Miller, M. D. et al., 1992, J. Exp. Med., 176: 1739-1744).
Perhaps the best studied of the antigen delivery systems are those derived from the linear polymeric esters of lactic acid and glycolic acid (i.e., poly (DL-lactide-co-glycolide)) (PLCG) (Edelman, R. et al., 1993, Vaccine, 11: 155-158; Eldridge, J. H. et al., 1989, Curr. Top. Microbiol. Immunol., 146: 59-66; Eldridge, J. H. et al., 1990, J. Controlled Release, 11: 205-214; Eldridge, J. H. et al., 1989, Adv. Exp. Med. Biol., 251: 191-202; Eldridge, J. H. et al., 1991, Mol. Immunol., 28: 287-294; Eldridge, J. H. et al., 1991, Infect. Immun., 59: 2978-2986; Marx, P. A. et al., 1993, Science, 260: 1323-1327; Moldoveanu, Z. et al., 1993, J. Infect. Dis., 167: 84-90; O""Hagan, D. T. et al., Vaccine, 11: 149-154; O""Hagan, D. T. et al., 1991, Immunology, 73: 239-242; Ray, R. et al., 1993, J. Infect. Dis., 167: 752-755; Reid, R. et al., 1993, J. Immunol., 150: 323A; Reid, R. H. et al., 1993, Vaccine, 11: 159-167). Encapsulation of putative antigens into PLCG microcapsules affords a number of advantages. First, microcapsules are easily degraded by hydrolysis to form lactic acid and glycolic acid. Second, PLCG microcapsules less than 5 xcexcm in size readily penetrate Peyer""s patches, mesenteric lymph nodes and spleen after oral inoculation of mice. Third, oral intraperitoneal, intranasal or subcutaneous inoculation of mice with PLCG microencapsulated antigens including influenza virus, parainfluenza virus, simian immunodeficiency virus, Staph. aureus enterotoxin B toxoid, and ovalbumin induces a greater immune response than that induced in animals inoculated with the same dose of free virus or protein. In addition, oral inoculation of mice with inactivated viruses induces an enhanced antigen-specific IGa response at mucosal surfaces. Lastly, PLCG microcapsules have been administered orally to adult volunteers without adverse effects.
The major disadvantage of PLCG microcapsules is the requisite use of organic solvents. Contact with organic solvents tends to inactivate the infectivity of viral and bacterial pathogens, and, in addition, may alter the immunogenicity of surface proteins critical to induction of humoral or cellular immune responses. In fact, large quantities of viral proteins have been required to induce an antigen-specific immune response with PLCG microcapsules.
U.S. Pat. No. 3,137,631 relates to encapsulation of water insoluble organic liquids by cross-linking synthetic resins through the application of heat or catalysts or both. The capsule shells are described as formed from covalently linked non-ionic materials or from heat denaturable proteins. The resultant capsules benefit from secondary treatment with cross-linking agents to impart increased stability to the capsule.
U.S. Pat. No. 4,205,060 discloses microcapsules comprising a core containing a water soluble salt formed by reaction between a polymeric ionic resin and a medicament, formed either by reaction of an acidic polymer with a basic medicament or, conversely, a basic polymer with an acidic drug. The walls of the microcapsules are formed from water-insoluble film-forming polymers. The water-insoluble film-forming polymers identified as suitable sheathing agents are all neutral non-ionized polymers. The capsules of that invention are made by preparing an aqueous solution of a salt made by reacting a medicament and a core polymer; preparing a solution of a water-insoluble sheath-forming polymer in a first water-immiscible organic liquid; dispersing the aqueous solution in the organic solution; and adding to the dispersion a second water-immiscible liquid which is a non-solvent for the sheath-forming polymer to precipitate the film around droplets of the dispersed aqueous phase.
U.S. Pat. No. 4,606,940 discloses the preparation of microcapsules by coacervation to precipitate the encapsulating material. A single colloid is dispersed in water and the water of salvation is removed from around the colloid by addition of chemical compounds which have a greater affinity for water than the colloid. This causes the colloid chains to come closer together and form the coacervate. Temperature changes are needed to facilitate the encapsulation by coacervation.
U.S. Pat. No. 3,959,457 discloses microcapsules comprised of the reaction product produced in a finely dispersed emulsion of a water-immiscible solution of (a) an organic polyfunctional Lewis base, in a (b) low boiling point, polar, organic solvent, and an aqueous solution of a (c) partially hydrophilic, partially lipophilic, polyfunctional Lewis acid. The capsules of that invention have lipophilic cores.
U.S. Pat. No. 5,132,117 discloses microcapsules that consist of aqueous or substantially aqueous cores surrounded by capsular anisotropic Lewis salt membranes. These aqueous-core microcapsules are prepared by dispersing an aqueous solution of a suitable Lewis-acid wall-forming reactant and a core material in a suitable non-aqueous solvent, adding an additional amount of non-aqueous solvent containing a suitable Lewis-base wall-forming reactant, and harvesting the microcapsules formed by the interfacial reaction. Alternatively, the aqueous-core microcapsules of that patent may be prepared by dispersing an aqueous solution of a suitable Lewis-acid wall-forming reactant and a core material in a suitable non-aqueous solvent containing a suitable Lewis-base wall-forming reactant and harvesting the microcapsules formed by the interfacial reaction.
F. Lim, in Belgium Patent No. 882,476, (1980), describes a process in which calcium alginate microspheres are first formed, then surface treated to convert them to poly-lysine or poly-ethylenimine alginate coacervates and finally core-liquified by treatment with a calcium chelating agent.
Rha and Rodriques-Sanchez, in U.S. Pat. No. 4,744,933 (1988), simplify the Lim procedure by spraying one charged polymer directly into an oppositely charged polymer to produce a complex coacervate similar to that of Lim.
Dautzenberg et al., in U.K. Patent Application 2 135 954 A (1984), similarly describe formation of complex coacervate microcapsules by forcing 2 to 3 mm droplets of anionic polymer solutions to fall several tens of centimeters into solutions of oppositely charged poly-quaternary ammonium salts. In all of these other methods, it is clear that high viscosity polymer solutions are required to produce microcapsules effectively, and all employ two oppositely charged polymers to form complex coacervates.
Ito et al., Science, 263:66-68 (1994) have used time lapse confocal laser micrographs to demonstrate the tendency toward inhomogeneity of colloidal solutions of anionic polymers, such as sodium polyacrylate, with the development of some microregions of relatively high polymer concentrations and other regions with no polymer.
The present invention provides microencapsulation technology analogous to that described above with reference to U.S. Pat. Nos. 3,959,457 and 5,132,117, but different in that it utilizes an all aqueous system. The microcapsules of this invention are based on formation of poorly soluble (amine) salts of polyanionic macromolecules. This process is capable of producing uniform size particles under very gentle conditions.
By contrast, many of the previously known entirely aqueous systems are based on formation of coacervates, either simple or complex, and provide microbeads of widely ranging particle size. B. R. Mathews and J. R. Nixon, Surface characteristics of gelatin microcapsules by scanning electron microscopy, J. Pharzm. Pharmacol. 26:383-384 (1974). Some simple coacervates suffer from the disadvantage of requiring strongly acid (e.g. pH 3-4) media to precipitate proteinaceous coacervates. Complex coacervates precipitated from aqueous solution require at least two oppositely charged polymers. Entirely aqueous systems for preparation of hydrogels based on hydroxyethylacrylate involve free radical polymerization catalyzed by per-oxy species or ionizing radiation. J. D. Andrade, D. Gough, B. Kolff, W. J. Kunitomo and R. V. Wagenon, Coated adsorbents for direct blood transfusion: HEMA/activated carbon, Trans. Amer. Soc. Artif. Int. Organs 17:222-228 (1971). Such catalysts are likely to be destructive of fragile protein molecules or intact organisms. It is known that hydrogels prepared from aqueous alginic acid and calcium ion can be made in a process gentle enough to embed and preserve live for later release both microbes and multicellular organisms (e.g., nematodes). F. Lim and A. M. Sun, Science 210:908-910 (1980). Moreover, the calcium alginate system appears to be limited to that single alginate salt, and would not provide the amine salts of the present invention.
A number of recent papers describe other means to encapsulate immunogenic materials but rely on non-aqueous systems. J. H. Eldridge et al. (1991) Molecular Immunology SUPRA., R. Edelman, et al. (1993) Vaccine SUPRA., and R. Reddy, S. Nair, K. Byrnestad and B. T. Rouse, Liposomes as antigen delivery systems in viral immunity. Sem. Immunol. 4:91-96 (1992). Immunogenic subunit vaccine components have been captured in poly-acrylate and poly-glycolide/lactide beads or liposome-like vesicles through processes utilizing volatile organic solvents such as dichloromethane or chloroform. The solvents are used to form emulsions of polymer solution or dried lipid films. Poly-acrylate and poly-glycolide/lactide processes typically result in microbeads with extremely low (approximating 0.01%) immunogen or antigen capture efficiency compared to the relatively higher (approximating 5%) efficiency seen in the present, not yet optimized, process.
Thus, there remains a need for effective systems for microencapsulation of active agents, and immunogenic substance in particular.
According to one aspect, the present invention provides stable microcapsules that have aqueous cores and are substantially free of non-aqueous contaminants. The microcapsules may advantageously comprise an active agent. The invention further provides a highly efficient method of preparing such microcapsules.
This invention also provides means for encapsulating materials using an entirely aqueous system of reagents at or below room temperature and without need for high pressures. As such, it has application to many substances or entities which are unstable to the organic solvents, elevated temperatures, and/or high pressures heretofore employed in most encapsulation systems. Most notable among such substances and entities are naturally occurring or biotechnologically derived enzymes, proteins and peptides such as glucose 6-phosphate dehydogenase, calcitonin, erythropoietin, hemoglobin, insulin, interleukin, or somatotropin, naturally occurring non-proteinaceous macromolecules such as heparin, vaccines and vaccine components derived from intact or immunogenic subunits including xe2x80x9cnakedxe2x80x9d desoxyribonucleic acid (DNA) and desoxyribonucleic acid constructs, and/or derived from intact or attenuated organisms or their immunogenic subunits including actinomyces, bacilli, cocci, fungi, helminths, larvae, prions, protozoa, rickettsia, spirochetes, viruses, multicellular parasites and yeasts, tolerizing antigens used for immunization against or attenuation of allergic responses to dusts, danders, pollens, spores and the like, and cells such as pancreatic islet cells, hepatocytes, interleukin- and other immunomoudulator-secreting cells derived from human or other species when implated to serve as surrogates for damaged, dysfunctional or missing tissues and/or organs which, if not encapsulated, might be recognized as foreign to the recipient organism and subject to unwanted immunologic attack.
This invention further provides means for encapsulating and later releasing highly irritant drugs, such as fluorouracil, at a rate slow enough to reduce the toxicity of such agents, as well as to encapsulate, release slowly, and sustain uniform therapeutic concentrations of numerous drugs (typified by anti-inflammatory agents such as prednisolone and indomethacin, antibodies such as tetracycline or antispasmodic drugs such as theophylline). When used to encapsulate pigmented or opaque materials such as blue dextran or charcoal, the system may be used to photoprotect bioactive agents such as ivermectin (an ectoparasiticide) and Bt proteins (Bacillus thuringiensis larvacidal proteins) which are unstable to light, and to release such agents either gradually or in triggered bursts. Fluorescently labelled microcapsules may be made and used to color code, identify, or aid in detecting and locating encapsulated formulations.
According to another aspect, the present invention provides encapsulated rotavirus particles, and other such agents which are typically unstable and/or denatured by organic solvents, elevated temperatures, and/or high pressures heretofore employed in most encapsulation systems. The rotavirus which are encapsulated according to the present invention include reassortant strains of rotavirus which are particularly useful as vaccines to protect against rotavirus infection.
As will appear from the following description, the present invention enables vaccine delivery in a way which allows for penetration of antigen into mucosal lymphocyte populations (e.g., Peyer""s patch) after oral inoculation, as well as persistence of antigen in tissues after oral or parenteral inoculation.